Chemoenzymatic Studies to Enhance the Chemical Space of Natural Products

Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products. Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most challenging part was to modify the sugar moiety. In order to solve this problem, PV was used as an alternative lead-structure for modification because its sugar moiety offered the possibility of enzymatic O-methylation. We produced four PV derivatives with different methylation patterns for cytotoxicity assays and provided important structure-activity-relationship information. Mithramycin (MTM) is the most prominent member of the aureolic acid type anticancer agents. Previous work in our laboratory generated three MTM analogues, MTM SA, MTM SK, and MTM SDK by inactivating the mtmW gene. We developed new MTM analogues by coupling many natural and unnatural amino acids to the C-3 side chain of MTM SA via chemical semi-synthesis and successfully made some compounds with both improved bioactivity and in vivo tolerance than MTM. Some of them were consequently identified as promising lead-structures against Ewing’s sarcoma. The potential of selectively generating novel MTM analogues led us to focus on a key enzyme in the biosynthetic pathway of mithramycin, MtmC. This protein is a bifunctional enzyme involved in the biosynthesis of TDP-D-olivose and TDP-D-mycarose. We clarified its enzymatic mechanisms by X-ray diffraction of several crystal complexes of MtmC with its biologically relevant ligands. Two more important post-PKS tailoring enzymes involved in the biosynthesis of the MTM side chains, MtmW and MtmGIV, are currently under investigation. This would not only give us insight into this biosynthetic pathway but also pave the way to develop potentially useful MTM analogues by engineered enzymes

Semi-Synthetic Mithramycin Derivatives with Anti-Cancer Activity

Mithramycin derivatives and their pharmaceutically acceptable salts are disclosed. The mithramycin derivatives can be used in the treatment of Ewing sarcoma or other cancer or neuro-disease associated with an aberrant erythroblast transformation- specific transcription factor

The carbon footprint of power-to-synthetic natural gas by photovoltaic solar powered electrochemical reduction of CO2

The search for more sustainable production and consumption patterns entails the integration of emerging edge-cutting technologies. Holistic studies are needed in order to accurately evaluate properly the environmental competitiveness of the suggested solutions. Among those alternatives, it has been suggested the utilisation of CO2 for the production of synthetic natural gas, the so-called Power-to-Gas (PtG) technology. In this work, we use the PtG technology to analyse the environmental rationality in terms of the carbon footprint (CF) of a Photovoltaic (PV) solar powered Electrochemical Reduction (ER) process for the utilisation of CO2 as carbon source for the production of CH4. This synthetic natural gas is ready to be injected into the transmission and distribution network. The raw materials for the process are a source of CO2 (mixed with different ratios of N2), H2O and electricity from PV solar. The separated products are CH4, C2H4, H2/CO, O2 and HCOOH. The reaction, separation/purification and compression stages needed to deliver commercially distributable products are included. Mass and energy balances were used to create a black-box model. The input to the model is the faradaic efficiency and cathodic potential of the best cathodesperforming at lab-scale (over 60% faradaic efficiency towards CH4). It was assumed that cathodes were long-lasting. The output of the model is the distribution of products (related to 1 kg of pure CH4) and the energy consumption at each of the aforementioned stages. The overall CF is then calculated as a function of the CF PV solar reference and the total energy consumption. The effect on the distribution of each stage to the total energy consumption of both the purity of the CO2 stream and the conversion of CO2 in the reactor was analysed. The results show that the principal contributor to the total energy consumption is the ER of CH4 across all CO2concentrations and conversions. When a CO2 conversion of 50% is chosen together with an inlet stream with a N2:CO2 ratio of 24, the electricity consumption of the process is between 2.6 and 6.2 times the minimum obtained for a reference ER reactor including the separation and compression of gaseous products (18.5 kWh kg−1 of CH4). The use of PV solar energywith low CF (14⋅10−3 kg kWh−1) allows the current lab-scale performers to even the CF associated with the average world production of natural gas when the valorisation of C2H4 is included (∼1.0 kg kg−1 of CH4).Authors gratefully acknowledge the funding provided by the State Research Agency, Ministry of Science, Innovation, and Universities (Spain) through the project CTQ2016-76231-C2-1-R

木棉根皮之化學成分研究

木棉(Bombax malabarica DC.)屬於木棉科(Bombacaceae)木棉屬(Bombax L.)，原產地在印度、馬來西亞、及菲律賓等地，引進臺灣後已本土化成為本島常見的大型落葉喬木。木棉的花、樹皮、及根部常用於民間用藥中，而過去的科學研究也顯示木棉含有許多具生物活性的化合物，基於對天然物研究的興趣，以及希望發掘具生物活性之成分，我們利用木棉根皮為材料，進行了一系列的分離、純化、及結構鑑定等工作。實驗過程中，首先將酒精萃取物依極性劃分為二氯甲烷、正丁醇、和水可溶的三部分，經活性試驗發現具抑制甲型葡萄糖水解酶 (α-glucosidase) 功能之成分集中在正丁醇可溶部分，因此以Sephadex LH-20、Lobar RP-18、及半製備型HPLC等對正丁醇可溶部分進行分離，最後得到六個不同類型的化合物，分別是shorealactone (1)、adenosine (2)、(-)-epicatechin 5-O-β-D-xylopyranoside (3)、2-C-[β-D- apiofuranosyl-(1 → 6)]-β-D-glucopyranosyl]-1, 3, 6-trihydroxy-7-methoxyxanthone (4)、3, 4, 5-trimethoxyphenol-1-(6-xylopyranosyl) glucopyranoside (5)、及4-hydroxy-3, 5-dimethoxybenzoic acid (6)。二氯甲烷可溶部分經過相似的分離程序也得到四個化合物，分別是lupeol (7)、β-sitosterol (8)、isohemigossypol-1-dimethyl ether (9)、及化合物10。上述化合物使用包括核磁共振儀(NMR)、質譜儀(MS)等儀器進行結構解析。這十個化合物中，我們針對從正丁醇部分分出的化合物及化合物10做了β-glucosidase assay，僅化合物1、3、4對α-glucosidase有弱抑制活性，其餘均無活性。此外，化合物1推測是由bis-stilbenoid及ascorbic acid經植物酵素催化合成，經資料庫搜尋，發現此化合物為相當少見的化合物，首次發表是在2003年從Shorea hemsleyana樹皮中分離出來，本實驗則是第二次發現該化合物。有趣的是，S. hemsleyana與B. malabarica在傳統植物分類中屬不同科，化合物1的發現意味這兩種植物在化學分類法中有著某種程度的關聯。除此之外，在仔細地分析核磁共振光譜後，發現化合物10為新天然物，其部份架構與isohemigossypol-1-dimethyl ether (9)相似。Bombax malabarica DC. (Bombacaceae), commonly known as silk cotton tree, is native to India, Malaya, and Philippines, and has been naturalized in Taiwan. Its flower, stem bark, and root are common materials for folklore medical use. Past studies had led to the isolation of some bioactive constituents. Being interested in the exploration of bioactive natural ingredients, we reinvestigated this plant.he EtOH extract of the root bark of B. malabarica was divided into fractions soluble in CH2Cl2, n-BuOH, and H2O by liquid-liquid partitioning. Through α-glucosidase assay, we observed that the n-BuOH-soluble fraction showed strong inhibitory effects. Therefore, n-BuOH-soluble fraction was chromatographed over Sephadex LH-20, Lobar RP-18, and semi-preparative HPLC to give six compounds. They are shorealactone (1), adenosine (2), (-)-epicatechin 5-O--D-xylopyranoside (3), 2-C-[β-D-apiofuranosyl-(1→ 6)]-β-D-glucopyranosyl]-1, 3, 6-trihydroxy-7- βmethoxyxanthone (4), 3, 4, 5-trimethoxyphenol-1-(6-xylopyranosyl) glucopyranoside (5), and 4-hydroxy-3, 5-dimethoxybenzoic acid (6). Four compounds, i.e., lupeol (7), β-sitosterol (8), isohemigossypol-1-dimethyl ether (9), and compound 10, were isolated from the CH2Cl2-soluble fraction in a similar manner. The structures of these compounds were elucidated based on elaborated spectroscopic analysis. The compounds isolated from the n-BuOH-soluble fraction and compound 10 had been assayed against α-glucosidase. Only compound 1, 3, and 4 showed weak inhibitory effects. Of these compounds isolated, shorealactone (1) is an adduct of bis-stilbenoid and ascorbic acid, and represents the second appearance of such compound from the natural sources, the first one being isolated from Shorea hemsleyana in 2003. Interestingly, both species belong to different plant families. This discovery may imply a certain relationship between S. hemsleyana and B. malabarica on the basis of chemical taxonomy. In addition, compound 10 is a new natural product, possessing an unsaturated moiety the same with isohemigossypol-1-dimethyl ether (9).Committee member verification (Chinese)………………………………………………icknowledgement (Chinese) …………………………………………………………...iibstract (Chinese)………………………………………………………………………iiibstract (English)………………………………………………………………………..vhapter 1. Introduction…………………………………………………………………..1.1 Objective of the research……………………………………………………….1.2 Introduction to B. malabarica…….…………………………………….. 3.3 Previous chemical investigation of B. malabarica……………………………..6.4 Introduction to type 2 diabetes………………………………………………..13.5 Introduction to α-glucosidase inhibitors……………………………………...18hapter 2. Results and Discussions…………………………………………………….21.1 Compounds isolated from the root bark of B. malabarica……………..21 2.2 Structure elucidation of compound 1………………………………………….24 2.3 Biosynthetic pathway of compound 1………………………………………...32 2.4 Structure elucidation of compound 2………………………………….............34 2.5 Structure elucidation of compound 3………………………………………….36 2.6 Structure elucidation of compound 4………………………………………….39 2.7 Structure elucidation of compound 5…………………………………….........45 2.8 Structure elucidation of compound 6………………………………………….50 2.9 Structure elucidation of compound 7………………………………………….51 2.10 Structure elucidation of compound 8………………………………………...55 2.11 Structure elucidation of compound 9………………………………………...59 2.12 Structure elucidation of compound 10……………………………………….64 2.13 Effect of compound 1 –6, 10 on α-glucosidase…………………………….71.14 Conclusions………………………………………………………………….73hapter 3. Experimental Section……………………………………………………….76 3.1 Apparatus and materials……………………………………………………….76 3.1.1 Apparatus………………………………………………………………..76 3.1.2 Solvents and reagents…………………………………………………77 3.1.3 Apparatus and reagents used on α-glucosidase assay…………………..78 3.2 Assay for α-glucosidase activity……………………………………………...79 3.2.1 Introduction……………………………………………………………..79 3.2.2 Enzyme activity unit…………………………………………………….79 3.2.3 Methods…………………………………………………………………79 3.2.4 Calculation of the IC50 values…………………………………………...82 3.3 Experimental procedure……………………………………………………….83 3.3.1 Extraction of the root bark of B. malabarica………………...83 3.3.2 Fractionation of n-BuOH-soluble fraction……………………………...83 3.3.2.1 Isolation of shorealactone (1), adenosine (2), (-)-epicatechin 5-O- β-D-xylopyranoside (3)………………………………………….83 3.3.2.2 Isolation of 2-C-[β-D-apiofuranosyl-(1 → 6)]-β-D-glucopyranosyl] -1, 3, 6-trihydroxy-7-methoxyxanthone (4)……………………...84 3.3.2.3 Isolation of 3, 4, 5-trimethoxyphenol-1-(6-xylopyranosyl) gluco- pyranoside (5)……………………………………………...84 3.3.2.4 Isolation of 4-hydroxy-3, 5-dimethoxybenzoic acid (6)…………84 3.3.3 Fractionation of CH2Cl2-soluble fraction……………………………….85 3.3.3.1 Isolation of lupeol (7) and β-sitosterol (8)……………………….85 3.3.3.2 Isolation of isohemigossypol-1-dimethyl ether (9).......................86 3.3.3.3 Isolation of compound 10………………………………………..86.4 Physical properties of compound 1–10...........................................................8

Electroless Nickel Deposition for Front Side Metallization of Silicon Solar Cells

In this work, nickel thin films were deposited on texture silicon by electroless plated deposition. The electroless-deposited Ni layers were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), X-ray diffraction analysis (XRD), and sheet resistance measurement. The results indicate that the dominant phase was Ni2Si and NiSi in samples annealed at 300–800 °C. Sheet resistance values were found to correlate well with the surface morphology obtained by SEM and the results of XRD diffraction. The Cu/Ni contact system was used to fabricate solar cells by using two different activating baths. The open circuit voltage (Voc) of the Cu/Ni samples, before and after annealing, was measured under air mass (AM) 1.5 conditions to determine solar cell properties. The results show that open circuit voltage of a solar cell can be enhanced when the activation solution incorporated hydrofluoric acid (HF). This is mainly attributed to the native silicon oxide layer that can be decreased and/or removed by HF with the corresponding reduction of series resistance

Designing a Thermal Radiation Oven for Smart Phone Panels

Thermal radiation is the only heat transfer mechanism with vacuum compatibility, and it carries energy at light speed. These advantages are taken in this work to design an oven for smart phone panels. The temperature of panels is acquired from a numerical method based on finite-difference method. The space configuration of the heating lamps as well as the relative distance between lamps and the panel are control factors for optimization. Full-factorial experiments are employed to identify the main effects from each factor. A fitness function Q considering both temperature uniformity of the panel and the heating capability of the ovens is proposed. The best oven among 27 candidates is able to raise panel temperature significantly with high uniformity

Rosiglitazone Suppresses In Vitro Seizures in Hippocampal Slice by Inhibiting Presynaptic Glutamate Release in a Model of Temporal Lobe Epilepsy.

Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg2+ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg2+ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10 μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10 μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE

Clinical Outcomes and Prolonged SARS-CoV-2 Viral Shedding in ICU Patients with Severe COVID-19 Infection and Nosocomial Bacterial Pneumonia: A Retrospective Cohort Study

Objectives: This study explored the clinical outcomes and association of prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding in patients with severe coronavirus disease 2019 (COVID-19) infection who developed nosocomial pneumonia. Methods: This was a retrospective study conducted in a medical center in Taiwan. From May to September 2021, patients from four intensive care units were enrolled after SARS-CoV-2 was confirmed through quantitative polymerase chain reaction and all cases were compatible with the definitions of severe COVID-19 infection. Baseline characteristics, disease severity, clinical outcomes, and times of viral shedding were recorded. Results: A total of 72 patients were diagnosed as having severe COVID-19 infection and 30 developed nosocomial pneumonia, comprising hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). The patients with severe COVID-19 infection and concomitant HAP/VAP had longer intensive care unit (ICU) stays and fewer ventilator-free days at Day 28. An independent risk factor for nosocomial pneumonia was a greater SOFA score at admission. Furthermore, the patients with severe COVID-19 infection who developed HAP/VAP had a significantly longer duration of SARS-CoV-2 shedding (19.50 days vs. 15.00 days, p = 0.006). Conclusions: Patients with severe COVID-19 infection who developed nosocomial pneumonia had longer SARS-CoV-2 shedding days, more complications, and worse outcomes